The long-term goal of this work is to understand how epigetically heritable silent chromatin domains are assembled and inherited. Silent domains are a conserved feature of eukaryotic chromosmes and play important roles in regulation of gene expression and maintenance of chromosome stability. The budding yeast Saccharomyces cerevisiae contains epigenetically heritable silent chromatin domains that are amenable to both genetic and biochemical analysis. These domains are assembled and maintained by the combined activities of nucleator proteins that bind to DNA and a silencing complex called the SIR (Silent Information Regulator) complex. The SIR complex is composed of the NAD-dependent deacetylase Sir2, the histone binding protein Sir3, and an adaptor protein Sir4. The nucleator proteins recruit the SIR complex to DNA. The SIR complex then spread along the chromatin fiber away from the nucleation sites in a deacetylation- dependent manner, creating a chromatin domain that silences transcription and other DNA transactions. Despite an increase in our knowledge of how the SIR complex is recruited to DNA and binds to chromatin, it has remained unclear how it silences the chromatin regions with which it is associated and how the resulting silent domains are epigenetically inherited during chromosome duplication and cell division. In this proposal we will use a combination of biochemical assays in a system that relies on in vitro reconstitution of silent chromatin to investigate (1) the molecular mechanism of SIR-mediates gene silencing and (2) the mechanism of epigenetic inheritance of silent chromatin. In addition, we will investigate how the SIR complex changes the structure of the underlying chromatin in order to understand how possible chromatin compaction contributes to silencing. These studies are aimed at a complete molecular understanding of gene silencing through the molecular dissection of in vitro reconstituted silent chromatin. The conservation of silent chromatin domains and Sir2-like deacetylases suggests that the principles developed here for the budding yeast complexes will apply in other settings. A basic understanding of the mechanism of gene silencing will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention. PUBLIC HEALTH RELEVANCE: Epigenetic regulatory mechanisms are widely conserved in eukaryotes and contribute to normal development and disease progression in humans. The conservation of silent chromatin domains and Sir2-like deacetylases suggests that the principles developed by our proposed studies for the budding yeast complexes will apply in other settings. A basic understanding of the mechanisms that mediate the function and epigenetic inheritance of silent chromatin will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.